Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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SYNERGI5TIC EFFECT OF LANTIBIOTICS
IN COMBINATION WITH SELECTED AGENTS
AGAINST GRAM NEGATIV~_BACTERIA
Background of khe Invention
Nisin, a member of the group of antimicrobials
known as lanthionine bacteriocins, is an antimicrobial
polypeptide produced by certain strains of Lactococcus
lactis (formerly Streptococcus lactis). It is
manufactured through the pure-culture fermentation of
these bacteria with subsequent purification and
dryingO M. Doyle in Food Technol. 1988 42: (4~
169-171, describes the inhibitory effects of nisin on
the growth and survival of L. monocytoqenes.
The term "lantibiotics" was coined by Schnell et
al. (1988 Nature 333: 276-273) to describe a group of
bacteriocins including nisin which contain the amino
acid lanthionine and other "non-protein" amino acids.
This class of bacteriocins includes subtilin, pep 5,
epidermin, gallidermin, cinnamycin, RoO9-0198,
duramycin and ancovenin. These ribosomally synthe-
sized peptide bacteriocins contain from 19 to 34
amino acids and are produced by various microbes
including Staphlococcus species, Bac~11us species and
Streptomyces species.
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The combination of nisin with other material~ to
enhance its antimicrobial activity has been reported.
Thus, International patent application WO 89J12399
discloses the combination of ni~in with chelating
agents and~or surfactants for use against Gram
positive and gram negative ~acteria.
Published European patent application 0,384,319
discloses the synergistic effect of nisin with
chelating agents against L. monocytoqenes. This
reference discloses the use of certain amino acids
and monocarboxylic acids in combination with nisin.
While numerous references describe synergistic
combinations of nisin in combination with other
ingredients as being effective against Gram positive
bacteria, the activity of nisin, e ther alone or in
combination with other materials against Gram negative
bacteria is not widely reported.
Summary of the In~ention
The present invention involves a method for
inhibiting the growth of Gram negative bacteria in an
environment where their growth is undesirable which
method comprise~ introducing to said envixonment a
synergistically effective combination of a lanthionine
barteriocin and a synergist therefore wherein said
synergist is selected from the group consisting of
amino acids, aliphatic mono- and di- carboxylic
organic acids containing from 1 to 8 carbon atoms or
a suitable alkali metal or alkaline earth me*al salt
thereof, phenolic antioxidant antimicrobials, ~enzoic
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or sorbic acid and a suitable alkali metal or alkaline
earth metal salts thereof.
Description of the Invention
The present invention relates to the observat,ion
tha~ a lanthionine bacteriocin in combination with
certain additives has provided a composition which
inhibits the growth of undesirable Gram negative
bacteria to a greater degree than either material by
itself and that the combined inhibitory effect of
these substances is greater ~han the additive effect
observed when using the lantibiotic or other compo-
sition individually.
Various classes of compositions which act
synergistically with the lantibiotics have been
id~ntified. Among these are amino acids and in
particular glycine.
Another class of agents which has been found to
produce a synergistic antimicrobial effect in combi-
nation with a lantibiotic are aliphatic mono- and di-
carboxylic acids containing from 1 to 8 carbon atoms.These acids include lactic, propionic, acetic, malic,
tartaric, succinic, maleic, oxalic and fumaric acid.
Suita~la salts, such as the alkali and alkaline earth
metal salts, e.g. sodium, potassium and calcium can
be employed. Sorbic acid and suitable salts thereof
also exhibit synergistic activity against Gram
negative bacteria when used in combination with a
lantibiotic such as nisin.
The mechanism of action of lantibiotics such as
nisin has not been resolved, but many investigators
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have linked ~he lethality of ~hese peptides to
irreversible changes in the cell membranes of sus-
ceptible organisms. The resiskance of some organisms
to nisin is thought to be due to the lack o~ access
of the peptide to its site of action, either because
of physical exclusion or the lack of appropriate
receptor molecules. The lantibiotic synergists of
the present invention appear to alleviate this
inhibition of the access to the taxget microorganism
although the mechanism of action i5 not understood.
The present invention is predicated on the discovery
that a lantibiotic and certain ~ynergists therefore,
in combination, inhibit the growth of undesirable
Gram negative bacteria to a grea~r extent than
either material by itself and that the combined
inhibitory effect of these substances is greater than
the additive effect obser~ed when using the lanti-
biotic or synergist individually.
The lantibiotics are generally regarded as being
ineffective against Gram negative bacteria in the
absence of a potentiator. In the previously mentioned
WO 89/12399, chelating agents and surfactants are
described as being suitable potentiating agents for
the control of Gram negative species. It has now
been discovered that amino acids and certain mono-
and di- carboxylic organic acids as well as sorbic
acid and salts thereof act as potentiators which
render lantibiotics such as nisin effective against
Gram negative bacteria. In a typical example, when
using a lantibiotic such as nisin and a synergist
therefore, the total amount of nisin will typically
range from about 0.1 IU/gm (or IU/ml in liquid
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systems) of the substrate material being treated up
to as much as 2,000 IU/gm or IU/ml in a liquid
system. In most applications, a nisin concen~ration
of from 50 to 500 IU/gm will be sufficient due to the
boost it will receive from the synergist. As used
herein, the term IU is intended to refer to Interna-
tional Unit as determined by the assay of Tramer and
Fowler in J. Sci~ Food and Agric., 15(8) 522 (1964).
The amount of synergist to be employed will depend on
the particular composition involved. In yeneral the
amount of synergist in terms of weight percent of the
substrate being treated will be within the ranges set
out in Table A. Lanthionine bacteriocins other than
nisin can be expected to be efficacious at these
concen~rations although some routine experimentation
may be necessary to determine the concentration of a
particular lantibiotic acid synergist therefore for
optimal synergistic effect.
TABLE A
20 Class of Range of Preferred
Synerqist Suqqested Use Ranqe
Amino Acids 0.001% - 5~ 0.03% - 0.1%
Aliphatic mono- 0.001~ - 25% 0.01% - 1
and di- carboxylic
25 organic acids
Sorbic Acid ~ Salts 0.001% 2% 0.02% - 0.3%
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Surface treatment of a target substrate can be
accomplished by suspending the material to be treated
in a lanthionine~synergist solution. Alternatively,
the material may be dipped in the solution or it can
be sprayed onto the surface of the material. The
lanthionineisynergist combination can be incorporated
into films or gels which are applied to the substrate's
surface or the combination can be incorporated
directly into the environment keing treated such as
by adding it to milk to be used in making cheese. In
film coating applications such as dips, films, gels
or casings, the initial concentrations of the lanti-
biotic and synergist can approach their solubility
limits to provide a finished product which, after
application and diffusion, contains residual levels
of lantibiotic/synergist which are within the desired
ranges. When treating the material's surface, some
routine experimentation may be needed to ascertain
the most effective concentra1ion.
Environments capable of being treated with the
compositions of the present :invention include, but
are not limited to, meats and meat products, such as
chicken, turkey, ham, beef, salami, sausages and
smoked meat products; mayonnaise; dairy products such
as cheese, milk and yogurt; oils, fish and fish
products; soft drinks including natural juices;
animal Eeeds and other high protein products. In
addition to use in Eoods, the compositions of the
present invention have non food uses such as inclusion
in mouthwashes, denture cleaners, ointments, creams
and shampoos.
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Amony the problematical Gram negative bacteria,
Salmonalla typhimurium is a particular pest because
-
of its ubiquitous sontamination of fresh poultry.
Other undesirable Gram negative bacteria whose growth
can be inhibited by the synergistic combinations of
the present invention include other species of
Salmonella such as enteriditis and species from the
-
genera Shiqella and Camylobacter as well as various
strains of Escherichia coli.
The presen~ invention is further illustrated by
the following examples in which International Units
of nisin are expresses as U nisin. Nisin for these
studies was prepared from Nisaplin~ (Aplin & Barrett)
by diafiltration and ultrafiltration resulting in a
preparation which is essentially salt free,
EXAMPLE I
The synergistic bacteriocidal activity of nisin
and glycine against Salmonel:La tvPhimurium ATCC 14028
was evaluated at pH 3.5, 4.5 and 5.5 in 10 mM citrate
buffer (C~B). Nisin was tested at 500 and 1000 IU/ml
while glycine concentrations were 6.4, 16, 40, 100
and 250 mM. Controls were run in CAB. All stock
solutions, culture dilutions and treatments were
prepared in CAB at the pH evaluated. Treatments were
prepared in 0.9 ml quantities in 1 ml microtubes.
The treatment time was initiated with the addition o
0.1 ml of S. ty~himurium in its logrithmic growth
phase at approximately 1 x 10' CFU/ml. The treatment
time was 60 minutes at 37C. Treatments were then
serially diluted in phosphate buffered saline (0.01
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M, pH 7.0) using microtiter plates. Enumeration of
surviving bacteria was done with tryptic soy agar
spread plates after 48 hours of incubation at 37C.
The results of this experiment are set out in Tables
1 and 2 where synergy is identi~isd with a "~" when
the bacteriocidal effects of the combination of test
compound and nisin was observed to be greater than
the additive log1O reductions of the individual
components of the combination followed by the amount
of the increase in bacteriocidal activity in terms of
"% over". The following calculation was used where T
= test compound alone, N = nisin alone and T + N =
combination.
loglO reduction T ~ N> (logl~ reduction T +
loglO reduction N) = synergy
For purposes of the above calculation, any
negative log reductions were assumed to be 0. Any
estimations where greater than (~) or less than (<)
are reported, exceeded the detection limits of the
system and where not used for calculation of synergy,
except in obvious cases, e.g. where the minimum
effect which could have occurred was synergistic.
The results of this experiment are summarized in
Table 1 where international units of nisin are
e~pressed as U nisin.
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TABLE 1
Ac~i~ity ~ nisin ln ~u~b~na~ion ~ith gly~ine (GLY)
ag~insS S_ 5YDr~ iY~ in 10 ~ ~i~xa~ buS~2r ~ ~elect~d p~'s
Tr~at~ent LoglO CFU/~l L~gl, syn2rgy/% ovar
U~l nisin/ reduction
llM GI.Y
pa 3.S
0/0 ~.30 __
0~6.4 5.34 -0.04
0 Otl6 ~.48 -O.la
0/4a 6.00 -0.70
0/100 6.34 -1.04
0/254 4.50 0.80
lOOOfO 3.95 1.35
1000/6.4 3.80 1.50 +/11.1
1000~16 4.81 0.49
1000/40 ~.54 0.76
1000/100 4.04 1.26
' 000/2~0 1.78 3.52 +/63.7
p~ 4.5
/ 7.45 __
0~6.4 7. ~3 0.02
0/16 7.48 -o . o~
0/40 7.49 -0.04
0/100 >7.48 ~-0.03
0/250 7.40 0.05
500/0 5.73 1.72
500/6.4 6.28 1.17
500/16 5.99 1.46
500/40 6.00 1.45
500/100 6.34 1.11
5~0~250 5. ~8 1.47
p~ 5.5
0/0 7.65 __
0/6.4 >7 ~ 48 <0.17
0~16 ~7.4a c0.17
0/40 7.53 0.12
- 0/100 7.36 0.29
0~250 7.38 0.27
500~0 3.08 4.57
500/6.4 3. ~0 4.35
500/16 3.23 4.42
5~0/~0 2.91 4.74 ~/1.07
500/100 ~ Z .80 4.85
500/250 2.34 5.31 +/9.7
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High concentrations of glycine were bacteriocidal
to S. t~phimurium at all three pH levels. Detectable
synergy with nisin occurred at pH 3.5 and 5.5.
EXAMPLE II
.
The inhib1tory effect of nisin and organic acids
against S. typhimurium was determined by the procedure
described in Example I.
The results of these experiments are set out in
Table 2 (lactic acid) and ~able 3 (propionic acid).
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TABLE 2
Ac~i~y o~ nisi~ a~ioYI 17ith 12cl:ic acid ~IA)
agz~ 3t ~-. S~=D~ in 10 ~ cit:ra~ b~2er zlt ~elected pll'~
~gl, CPlJ/ml I.og,O Syn~rgy/% over
Il/~ in/ redllctior
p~ 3.S
0/0 7.08 __
0~6.4 ~.2~ -0.15
0 0/16 6.81 0.27
0/40 6.15 0.93
0/100 ~2.00 >5.08
0/250 ~2.00 ~.08
1000/0 6.63 0~45
1000/6.4 3.53 3.55 ~/689
1000/'6 6.36 0.72
1000~0 ~.53 1.55 +/12.3
1000/'~0 ~2.00 >5.08
1000~250 ~2.00 >5.0a
p~ 4.5
/ 7.20 --
0/6.4 7.45 -0.25
0/16 7.26 -0.06
0/40 ~.43 -0.23
0/100 7.4a -0.28
0/250 7.28 -0.08
500/0 6.45 0.75
500/6.4 6.56 0.64
500/16 6.60 0.60
500/40 >7.00 <0.2
500/100 ~7.00 <0.2
500/250 >7.00 <0.2
p~ 5.5
0/0 7.34 __
0/~ . 4 ~.35 -0.02
0/16 7 .4a -0.14
0~40 7.49 -0.15
0/100 7.36 -0.02
0/2S0 7.45 -0.11
4~ 50~0 4.68 2.66
50/6.4 3.g7 3.37 ~/26.7
S0/16 3.79 3.55 +/33~4
50/40 4~34 3.00 ~/12.8
50/100 ~7,00 ~0,3~
50/250 >7.00 ~0.34
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TABLE 3
~ Ct~Yity 0~ n~in i~ ~o~bination ~th propio~ic acid (PAl
ag~nst S~ iY~d~ in 10 ~ cit~ate bufr~r a~ ~elected p~'s
Tr~atment Lag,~ CF~/~l LoqID Syneryy/% ovor
~/~1 ni~in/ r~duction
mH P~
~
p~ 3.5
0/0 6.95 __
Of6.4 6.79 0.16
0/16 6.~6 0.09
0/40 6.99 -0.04
0/lOo 6.20 0.75
0/2S0 ~2.00 >4.95
1000/0 6.40 0.55
1000/6.4 6.29 0.56
1000~16 6.13 0.82 +/28.1
1000/40 5.89 1.06 +/9.27
1000/100 4.g8 1.97 -/79.
1000/250 ~1.00 ~5.95
~0 p~ 4.5
0/0 ~.26 --
0/6.4 7.22 0.04
0/16 7.29 -0.03
0~40 7.28 -0.02
2X 0/100 7.41 -0.15
0/250 7.31 0.05
500/0 6.50 0.76
500/6.4 6.g3 0.73
500/16 6.59 0.67
500/40 >6.00 ~1.26
500~100 >6.00 <1.26
500/250 >6.00 <1.26
p~ 5.5
0/0 7.~2 __
0/6.4 7.43 -0.01
o/16 7.46 -0.04
0/40 ?.53 -0.11
0/100 7.5g -0.17
0/Z50 7.46 -0.04
4a 500/0 4.55 2087
500/6.4 5~11 2.31
500/16 6.34 1.08
500/40 >6.00 <1.42
00~100 >6.00 <1.42
S00/Z50 >6.0~ <1.~2
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The higher concentratLons of lactic and propionic
acids alone were bacteriocidal to the test organism
a~ pH 3.5. Synergy with lactic acid occurred at all
pH levels evaluated at concentrations between 6.4 and
40 mM while propionic acid/nisin synergy occurred
only at pH 3.5.
In general monocarboxylic acids of 3-8 carbon
atoms, i.e. propionic, butyric, pentanoic, hexanoic,
heptanoate and octanoate can bs us~d in conjunction
with lantibiotic in order to enhance its effectiveness
in controlling Gram negative bacteria.
EXAMPLE I I I
The activity of nisin and potassium sorbate
against S~ typhimurium was determined as described in
Example I. The results of this experiment are
summarized in Table 4. The results of this experiment
are set out in Table 4.
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TABLE 4
A~tivity ~ ni~in in ~GmbiD~tia~ ~i~h po~a~iu~
~xb~ K~qORB~TE) ~ga~ns~ S_ $Y5 dh~l UI} in 10 ~ ~itr~t~ burrer
at s~l~ted p~'s
Tr~tEe~t L~g~O C~l L~gl. Syn~rqy~% ov~r
~/~1 nisin/ reduction
~+gO~13Aq`E
P~ 3.~
0/0 6.91 __
0 0~0.00256 6.96 -0,05
0/0.0064 6.g2 -0.01
0/0.016 7.00 -o.og
0/0.04 6.47 0.44
0~0.1 ~2.00 >4.91
100/0 6.~8 0.23
100/O.OOZ56 6.65 0~26 +/13
100/0.0064 6.61 0.30 +/30.4
100/0.016 6.69 0.22
LOO/0.04 6.12 0.79 +/243
100/0.1 3.45 3.46
P~ ~.5
0/0 7.01 __
0/0.002~6 7.05 _0,04
o/0.00~4 7.1g -0.14
0/0.016 7.21 -0.20
0/0.04 7.21 -0.20
0/0.1 7.23 -0.22
50~0 6.~1 0.20
SO/0.00256 6.75 0.26 +/30.0
50~0.0064 >7.00 <1.00
50/0.016 6.81 0.20
50/0.04 >7.00 <1.00
50/0.1 >7.00 ~1.00
p~ 5.5
0/0 7.S8 --
O/O.nO256 7.38 0~20
0~0.0064 6.86 0.72
0/0.016 7.17 0.41
0~0.04 7.26 0.32
0/0.1 7.26 0.32
50/0 >7.00 ~0.5~
50/0.00256 >7.00 ~0.58
50/0.0064 >7.00 <0.58
50/0.016 >7.00 <0.58
50/0.04 >7.00 ~0.58
50/0.1 >7.00 <0.58
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Potassium sorbate by itself was bacteriocidal at
pH 5.5. Synergism with nisin was observed at various
concentrations at pH 3.5 and 4.5. Data at pH 5.5 was
inconclusive.
The pH range where a particular synergist is
effective will depend upon the environment being
considered. Thus, while a specific synergist may
show activity at a particular pH range in a buffer
system, a different pH range may be optimal in
certain food systems. For this reason, a certain
amount of routine experimentation may be necessary in
order to determine the optimal pH range for use of a
lantibiotic/synergist system in a particular envi-
ronment being treated.
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